Controlling metabolic pathways in plants has long been a goal of horticulturists. See, e.g. Bonner and Varner (1976) Plant Biochemistry, Academic Press, New York, which is incorporated herein by reference. The advent of recombinant DNA technology has provided new approaches to reaching that goal. While significant progress has been made in understanding gene regulation, control of plant gene regulation remains at a relatively early state of development.
The water soluble pigment flavonoids are significant in their contribution to the coloration and other properties of higher plants. For example, the flavonoids are responsible for most orange, scarlet, crimson, mauve, violet and blue colors, and contribute significantly to yellow, ivory and cream colored flowers. See, Harborne, (1976) Chemistry and Biochemistry of Plant Pigments, 2d ed., Goodwin (Ed.) Acad. Press, London. The most important of the pigment molecules are the anthocyanins, particularly pelargonidin, cyanidin and delphinidin. These are the darker colored pigments responsible for the orange-red, magenta and mauve colors, respectively. The other major flavonoid types, the chalcones, isomeric flavanones, flavones and flavonols are light colored and tend to have relatively smaller effects on intensity or patterns of color.
The functions of these pigments extend well beyond coloration of flowers, however. The pigments also color fruits, leaves and other plant parts, and importantly provide plants with UV protection, as well as protection against herbivores and microbes. Other uses include allelopathy and even some pharmaceutical applications.
The biosynthetic pathways of these various pigments have been extensively studied in many different plant species. The chalcones and aurones are products requiring only the initial biosynthetic enzymes, being direct products of the earliest precursors. The flavones and flavonols are intermediate, and the anthocyanins are products requiring substantial modifications from the initial precursors. All of these products are dependent upon the activity of the initial enzyme chalcone synthase (CHS), which catalyses the production of chalcone from three molecules of malonyl-Coenzyme A and one molecule of coumaroyl-Coenzyme A.
Essentially, all of these phenotypic traits have naturally evolved coordinately with constraints related to plant reproduction. For example, the appearance of a flower has generally resulted from the requirement to attract insects who assist in the pollination process essential for the sexual reproduction of the higher plants. Of course, the decorative and ornamental features impart to flowers a significant commercial value.
Mankind has traditionally intervened in some of the natural processes by, e.g., simply selecting particular flower colors and patterns which might otherwise not have survived in nature. Breeders routinely generate new and unusual flower phenotypes by a variety of time-tested breeding methods. The classical techniques for breeding improved plants, such as different flower varieties with altered flower color intensities or color patterns, typically required natural genetic variability within the experimental gene pool of the species and its relatives. More recently, the generation of variability by induction of mutations has been utilized. Breeders then select among the resulting population those products exhibiting interesting phenotypes, for further characterization.
Unfortunately, the induction of mutations to generate diversity often involves chemical mutagenesis, radiation mutagenesis, tissue culture techniques, or mutagenic genetic stocks. These methods provide means for increasing genetic variability in the desired genes, but frequently produce deleterious mutations in many other genes. These other traits may be removed, in some instances, by further genetic manipulation (e.g., backcrossing), but such work is generally both expensive and time consuming. For example, in the flower business, the properties of stem strength and length, disease resistance and maintaining quality are important, but often initially compromised in the mutagenesis process.
As noted, the advent of recombinant DNA technology has provided horticulturists with additional means of modifying plant genomes. While certainly practical in some areas, to date genetic engineering methods have had limited success in modifying the flavonoid biosynthetic or other pathways. Recently, the inhibition of flower pigmentation with a constitutively expressed "anti-sense" chalcone synthase gene has been reported (Van der Krol et al., (1988) Nature 333:866-869).
Thus, there exists a need for improved methods for producing plants with desired phenotypic traits. In particular, these methods should provide general means for phenotypic modification, and may lessen or eliminate entirely the necessity for performing expensive and time-consuming backcrossing.